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Description of key information

The test substance was evaluated in guinea-pigs according to the maximization method of Magnusson and Kligmanand to OECD No. 406, and EC Method B.6 guidelines. Under the experimental conditions and according to the maximization method of Magnusson and Kligman, the test substance is not a skin sensitizer.

 

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1999-03-09 until 1999-04-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Adequate data from an guinea pig maximisation test is available.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River France, 76410 Saint-Aubin-lès-Elbeuf, France
- Age at study initiation: approximately 3 months old
- Weight at study initiation: 364 ± 13 g for the males and 354 ± 15 g for the females
- Housing: the animals were housed individually in polycarbonate cages (48 cm x 27 cm x 20 cm) equipped with a polypropylene bottle
- Diet: "106 pelleted diet" (UAR, 91360 Villemoisson-sur-Orge, France).
- Water: drinking water filtered by a FG Millipore membrane (0.22 micron) was provided ad libitum
- Acclimation period: at least 5 days before the beginning of the study

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ±2°C
- Humidity: 30 to 70%
- Air changes: approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod: 12 h/12h

IN-LIFE DATES:from 1999-03-04 to 1999-04-12
Route:
intradermal and epicutaneous
Vehicle:
corn oil
Concentration / amount:
Induction: the test substance at a concentration of 5 % (w/w) in corn oil (intradermal injection) and the test substance at a concentartion of 25 % (w/w) in corn oil (topical application)
Challenge: first topical application- the test substance at the concentration of 10 % (w/w) in corn oil. Second topical application- the test substance at the concentration of 2 % (w/w) in corn oil.
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
Induction: the test substance at a concentration of 5 % (w/w) in corn oil (intradermal injection) and the test substance at a concentartion of 25 % (w/w) in corn oil (topical application)
Challenge: first topical application- the test substance at the concentration of 10 % (w/w) in corn oil. Second topical application- the test substance at the concentration of 2 % (w/w) in corn oil.
No. of animals per dose:
treated group: ten males and ten females
control group: five males and five females
Details on study design:
RANGE FINDING TESTS:
By intradermal route:
. 24 hours before treatment, the dorsal region of the animals was clipped,
. intradermal administrations of the test substance formulation (0.1 mL) at different concentrations were performed in the interscapular region,
. cutaneous reactions were evaluated approximately 24, 48 hours and 6 days after the injections.
By cutaneous route:
. 24 hours before treatment, both flank regions of the animals were clipped,
. a volume of 0.5 ml of the test substance formulation at the chosen concentrations was placed on a dry gauze pad (approximately 4 cm2) which was then applied to the skin and held in place by an occlusive dressing for 24 hours,
. cutaneous reactions were evaluated approximately 24 and 48 hours after removal of the dressings.

Criteria for selection of concentrations
The following criteria were used:
. the concentrations should be well-tolerated systemically and locally,
. intradermal injections should cause moderate irritant effects (no necrosis or ulceration of the skin),
. cutaneous application for the induction should cause at most weak or moderate skin reactions or be the maximal practicable concentration,
. cutaneous application for the challenge phase should be the highest concentration which does not cause irritant effects.


MAIN STUDY
A. INDUCTION EXPOSURE
Intradermal route:
On day 1, six injections were made deep into the dermis of a 4 cm x 2 cm clipped interscapular area, using a needle (diameter: 0.5 x 16 mm) mounted on a 1 mL plastic syringe (0.01 mL graduations).
Three injections of 0.1 ml were made into each side of this interscapular region as follows:
Anterior: FCA diluted at 50 % (v/V= with 0.9 % NaCl and the same in the control group
Middle: test substance at 5 % (w/w) in corn oil. In the control groul only vehicle
posterior: test substance at 5 % (w/w) in a mixture FCA /0.9 % NaCl 50/50 (v/v). The same in the control group but with vehicle instead of test substance.
The anterior and middle pairs of injections were performed close to each other and nearest the head, while the posterior pair was performed towards the caudal part of the test area.

Cutaneous route:
On day 7, the intrascapular area was clipped.
As the test substance was shown to be non-irritant during the preliminary test, the animals were treated with 0.5 mL of sodium lauryl sulphate (10%, w/w) in vaseline on order to induce local irritation.
On day 8, a cutaneous application to the region of the intradermal injections (4 cm x 2 cm) was performed as follows:

Control group: application of 0.5 mL of the vehicle.
Treated group: application of 0.5 mL of the undiluted test substance.

The test substance or the vehicle was placed on a dry gauze pad, which was then applied to the interscapular region.
The pad was held in place for 48 hours by means of an adhesive hypoallergenic dressing and an adhesive an allergenic waterproof plaster.
On removal of the dressing, no residual test substance was observed.
Cutaneous reactions were recorded 1 hour after removal of the occlusive dressing.


B. CHALLENGE EXPOSURE
First challenge applivation: on day 22, the animals of both groups received an application of 0.5 mL of the undiluted test substance to the posterior right flank and 0.5 mL of the vehicle to the posterior left flank. This application was performed using a 1 mL plastic syringe (0.01 mL) graduations). The test substance or the vehicle was placed on a dry gauze pad, which was then applied to 4 cm² (2 cm x 2 cm) clipped area of the skin.

The pads were held in contact with the skin for 24 hours by means of an occlusive, hypoallergenic dressing and an adhesive an allergenic waterproof plaster.
On removal of the dressing, no residual test substance was observed.

Second challenge application: on day 32, the animals of both groups received an application of 0.5 ml of the test substance at the concentration of 2 % (w/w) to the posterior left flank and 0.5 mL of the vehicle to the posterior right flank, under the same experimental conditions as for the first challenge application.
On removal of the dressing, no residual test substance was observed.
Challenge controls:
Not applicable
Positive control substance(s):
yes
Remarks:
, 2,4-Dinitro Chlorobenzene (DNCB)
Positive control results:
Under this experimental conditions and according to the Magnusson and Kligman method, the test substance DNCB at the concentration of 1 % (w/w) induced positive skin sensitization reactions in 90 % of the guinea-pigs.
Under this experimental conditions and according to the Magnusson and Kligman method, the test substance MERCAPTOBENZOTHIAZOLE at the concentration of 20 % (w/w) induced positive skin sensitization reaction in 30 % of the guinea-pigs.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
10 %
No. with + reactions:
19
Total no. in group:
20
Clinical observations:
well-defined or moderate erythema
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 10 %. No with. + reactions: 19.0. Total no. in groups: 20.0. Clinical observations: well-defined or moderate erythema.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
10 %
No. with + reactions:
13
Total no. in group:
20
Clinical observations:
well-defined or moderate erythema
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 10 %. No with. + reactions: 13.0. Total no. in groups: 20.0. Clinical observations: well-defined or moderate erythema.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Induction with no test substance and challenge as in the test group (10 % test substance)
No. with + reactions:
5
Total no. in group:
10
Clinical observations:
erythema
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: Induction with no test substance and challenge as in the test group (10 % test substance). No with. + reactions: 5.0. Total no. in groups: 10.0. Clinical observations: erythema.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Induction with no test substance and challenge as in the test group (10 % test substance)
No. with + reactions:
3
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: Induction with no test substance and challenge as in the test group (10 % test substance). No with. + reactions: 3.0. Total no. in groups: 10.0.
Key result
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
2 %
No. with + reactions:
20
Total no. in group:
20
Clinical observations:
a very slight or well-defined erythema
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: test group. Dose level: 2 %. No with. + reactions: 20.0. Total no. in groups: 20.0. Clinical observations: a very slight or well-defined erythema.
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
2 %
No. with + reactions:
3
Total no. in group:
20
Clinical observations:
a very slight or well-defined erythema
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: 2 %. No with. + reactions: 3.0. Total no. in groups: 20.0. Clinical observations: a very slight or well-defined erythema.
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
Induction with no test substance, challenge as in the test group (10 % test substance) and rechallenge with 2 % test substance
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
a very slight or well-defined erythema
Remarks on result:
other: see Remark
Remarks:
Reading: rechallenge. . Hours after challenge: 24.0. Group: negative control. Dose level: Induction with no test substance, challenge as in the test group (10 % test substance) and rechallenge with 2 % test substance. No with. + reactions: 10.0. Total no. in groups: 10.0. Clinical observations: a very slight or well-defined erythema.
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
Induction with no test substance, challenge as in the test group (10 % test substance) and rechallenge with 2 % test substance
No. with + reactions:
6
Total no. in group:
10
Clinical observations:
a very slight or well-defined erythema
Remarks on result:
other: see Remark
Remarks:
Reading: rechallenge. . Hours after challenge: 48.0. Group: negative control. Dose level: Induction with no test substance, challenge as in the test group (10 % test substance) and rechallenge with 2 % test substance. No with. + reactions: 6.0. Total no. in groups: 10.0. Clinical observations: a very slight or well-defined erythema.
Reading:
1st reading
Group:
positive control
Dose level:
0.1%
No. with + reactions:
9
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
0.1%
No. with + reactions:
7
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions and according to the maximization method of Magnusson and Kligman, the test substance is not a skin sensitizer.
Executive summary:

The test substance was evaluated in guinea-pigs according to the maximization method of Magnusson and Kligman and to OECD No. 406, and EC Method B.6 guidelines. Thirty guinea-pigs were allocated to two groups: a control group 1 (five males and five females) and a treated group 2 (ten males and ten females).

On day 1, intradermal injections of Freund's complete adjuvant mixed with the test substance (treated group) or the vehicle (control group) were performed in the interscapular region. On day 7, the same region received a topical application of sodium lauryl sulfate in vaseline (10%, w/w) in order to induce local irritation. On day 8, the test substance (treated group) or the vehicle (control group) was applied to the same test site which was then covered by an occlusive dressing for 48 hours. On day 22, after a rest period of 12 days, all animals of the treated and control groups were challenged by a cutaneous application of the test substance to the right flank. The left flank served as control and received the vehicle only. Test substance and vehicle were maintained under an occlusive dressing for 24 hours. Skin reactions were evaluated approximately 24 and 48 hours after removal of the dressing.

As equivocal cutaneous reactions were noted, a second challenge application was performed under the same experimental conditions after a rest period of 9 days, except that the test substance and the vehicle were applied to the left and right flanks, respectively.

Test substance concentrations were as follows:

Induction (treated group)

- intradermal injections: test substance at concentration of 5 % (w/w) in corn oil

- topical application: test substance at concentration of 25 % (w/w) in corn oil.

First challenge (all groups)

- topical application: test substance at a concentration of 10 % (w/w) in corn oil.

Second challenge (all groups)

- topical application: test substance at a concentration of 2 % (w/w) in corn oil

Skin samples were taken from the challenge application sites of all the animals. No histological examination was performed.

The sensitivity of the guinea-pigs was checked with a positive sensitizer, 2,4-Dinitro Chlorobenzene (DNCB). During the induction period, the reference substance DNCB was applied at the concentrations of 0.1% (w/w) (day 1) and 1% (w/w) (day 8) in com oil. For the challenge application, the reference substance DNCB was applied at the concentration of 1 % (w/w) in corn oil.

Results

After the first challenge application, a very slight or well-defined erythema was noted in 5/10 animals of the control group and a very slight, well-defined or moderate erythema, sometimes together with dryness of the skin, was observed in 19/20 animals of the treated group.

After the second challenge application, a very slight or well-defined erythema was noted in all animals of both groups at the 24-hour reading; these cutaneous reactions, sometimes together with dryness of the skin, persisted in a few animals of both groups at the 48-hour reading.

As the observed cutaneous reactions were of similar intensity, incidence and duration in the animals of the control and treated groups, and as the preliminary assays showed that the test substance is a slight irritant, these cutaneous reactions were most probably attributable to the irritant properties of the test substance (not subject of classification) and not to delayed contact hypersensitivity.

The species and strain which were used showed a satisfactory sensitization response in 90% animals treated with DNCB.

Conclusion

Under the experimental conditions and according to the maximization method of Magnusson and Kligman, the test substance is not a skin sensitizer.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The test substance was evaluated in guinea-pigs according to the maximization method of Magnusson and Kligman and to OECD No. 406, and EC Method B.6 guidelines. Thirty guinea-pigs were allocated to two groups: a control group 1 (five males and five females) and a treated group 2 (ten males and ten females).

On day 1, intradermal injections of Freund's complete adjuvant mixed with the test substance (treated group) or the vehicle (control group) were performed in the interscapular region. On day 7, the same region received a topical application of sodium lauryl sulfate in vaseline (10%, w/w) in order to induce local irritation. On day 8, the test substance (treated group) or the vehicle (control group) was applied to the same test site which was then covered by an occlusive dressing for 48 hours. On day 22, after a rest period of 12 days, all animals of the treated and control groups were challenged by a cutaneous application of the test substance to the right flank. The left flank served as control and received the vehicle only. Test substance and vehicle were maintained under an occlusive dressing for 24 hours. Skin reactions were evaluated approximately 24 and 48 hours after removal of the dressing.

As equivocal cutaneous reactions were noted, a second challenge application was performed under the same experimental conditions after a rest period of 9 days, except that the test substance and the vehicle were applied to the left and right flanks, respectively.

Test substance concentrations were as follows:

Induction (treated group)

- intradermal injections: test substance at concentration of 5 % (w/w) in corn oil

- topical application: test substance at concentration of 25 % (w/w) in corn oil.

First challenge (all groups)

- topical application: test substance at a concentration of 10 % (w/w) in corn oil.

Second challenge (all groups)

- topical application: test substance at a concentration of 2 % (w/w) in corn oil

Skin samples were taken from the challenge application sites of all the animals. No histological examination was performed.

The sensitivity of the guinea-pigs was checked with a positive sensitizer, 2,4-Dinitro Chlorobenzene (DNCB). During the induction period, the reference substance DNCB was applied at the concentrations of 0.1% (w/w) (day 1) and 1% (w/w) (day 8) in com oil. For the challenge application, the reference substance DNCB was applied at the concentration of 1 % (w/w) in corn oil.

Results

After the first challenge application, a very slight or well-defined erythema was noted in 5/10 animals of the control group and a very slight, well-defined or moderate erythema, sometimes together with dryness of the skin, was observed in 19/20 animals of the treated group.

After the second challenge application, a very slight or well-defined erythema was noted in all animals of both groups at the 24-hour reading; these cutaneous reactions, sometimes together with dryness of the skin, persisted in a few animals of both groups at the 48-hour reading.

As the observed cutaneous reactions were of similar intensity, incidence and duration in the animals of the control and treated groups, and as the preliminary assays showed that the test substance is a slight irritant, these cutaneous reactions were most probably attributable to the irritant properties of the test substance (which is not subject to classification) and not to delayed contact hypersensitivity.

The species and strain which were used showed a satisfactory sensitization response in 90% animals treated with DNCB.

Conclusion

Under the experimental conditions and according to the maximization method of Magnusson and Kligman, the test substance is not a skin sensitizer.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the results obtained from testing tert-butyl peroxyisobutyrate was not classified for skin sensitisation according to Regulation (EC) No 1272/2008 (CLP).